This invention is directed to assay devices for determination of characteristics of samples, unitized housings, and methods of determining the characteristics of samples using the assay devices.
Among the many analytical systems used for detection or determination of analytes, particularly analytes of biological interest, are chromatographic assay systems. Among the analytes frequently assayed with such systems are:
(1) hormones, such as human chorionic gonadotropin (hCG), frequently assayed as a marker of human pregnancy, as well as luteinizing hormone (LH), thyroid stimulating hormone (TSH), and follicle stimulating hormone (FSH); PA1 (2) antigens, particularly antigens specific to bacterial, viral, and protozoan pathogens, such as Streptococcus, hepatitis virus, and Giardia; PA1 (3) antibodies, particularly antibodies induced as a result of infection with pathogens, such as antibodies to the bacterium Helicobacter pylori and to human immunodeficiency virus (HIV); PA1 (4) other proteins, such as hemoglobin, frequently assayed in determinations of fecal occult blood, an early indicator of gastrointestinal disorders such as colon cancer; PA1 (5) enzymes, such as aspartate aminotransferase, lactate dehydrogenase, alkaline phosphatase, and glutamate dehydrogenase, frequently assayed as indicators of physiological function and tissue damage; PA1 (6) drugs, both therapeutic drugs, such as antibiotics, tranquilizers, and anticonvulsants, and illegal drugs of abuse, such as cocaine, heroin, amphetamines, and marijuana; and PA1 (7) vitamins. PA1 (1) a first opposable component including: PA1 (2) a second opposable component including a sample application zone. PA1 (1) a first opposable component including: PA1 (2) a second opposable component having a sample application zone. PA1 (1) a first opposable component including: PA1 (2) a second opposable component including a sample application zone. PA1 (1) a first opposable component including: PA1 (2) a second opposable component having a sample application zone. PA1 (1) a first opposable component including: PA1 (2) a second opposable component including a sample application zone. PA1 (1) a first opposable component including: PA1 (2) a second opposable component having a sample application zone.
Among the most important of such systems are the "thin-layer" systems in which a solvent moves across a thin, flat, absorbent medium. Among the most important of tests that can be performed with such thin-layer systems are immunoassays, which depend on the specific interaction between an antigen or hapten and a corresponding antibody or other specific binding partner. The use of immunoassays as a means for testing for the presence or amount of clinically important molecules has been known for some time. As early as 1956, J. M. Singer reported the use of an immune-based latex agglutination test for detecting a factor associated with rheumatoid arthritis (J. M. Singer et al., Am. J. Med., 22:888-892 (1956)).
Among the chromatographic techniques used in conjunction with immunoassays is a procedure known as immunochromatography. In general, this technique uses a disclosing reagent or particle that has been linked to an antibody to the molecule to be assayed, forming a conjugate. This conjugate is then mixed with a specimen and, if the molecule to be assayed is present in the specimen, the disclosing reagent-linked antibodies bind to the molecule to be assayed, thereby giving an indication that the molecule to be assayed is present. The disclosing reagent or particle can be identifiable by color, magnetic properties, radioactivity, specific reactivity with another molecule, enzymatic activity, or another physical or chemical property. The specific reactions that are employed vary with the nature of the molecule being assayed and the sample to be tested.
Immunochromatographic assays fall into two principal categories: "sandwich" and "competitive," according to the nature of the antigen-antibody complex to be detected and the sequence of reactions required to produce that complex. In general, the sandwich immunochromatographic procedures call for mixing the sample that may contain the analyte to be assayed with antibodies to the analyte. These antibodies are mobile and typically are linked to a label or disclosing reagent, such as dyed latex, a colloidal metal sol, a radioisotope, or an enzyme producing a detectable product. This mixture is then applied to a chromatographic medium containing a band or zone. This band or zone contains immobilized antibodies to the analyte of interest. The chromatographic medium is often in the form of a strip resembling a dipstick. When the complex of the molecule to be assayed and the labeled antibody reaches the zone of the immobilized antibodies on the chromatographic medium, binding occurs and the bound labeled antibodies are localized at the zone. This indicates the presence of the molecule to be assayed. This technique can be used to obtain quantitative or semi-quantitative results.
Examples of sandwich immunoassays performed on test strips are described by U.S. Pat. No. 4,168,146 to Grubb et al. and U.S. Pat. No. 4,366,241 to Tom et al., both of which are incorporated herein by this reference.
In a competitive immunoassay, typically, a labeled analyte or analyte analogue is supplied, and a competitive reaction is set up between the unlabeled analyte in the sample and the labeled analyte or analyte analogue for binding to an immobilized specific binding partner immobilized on the test strip. In general, competitive immunoassays are more suitable for assay of haptens, because they do not require the formation of a ternary sandwich complex.
Although useful, currently available chromatographic techniques using test strips have a number of drawbacks. Many samples, such as fecal samples, contain particulate matter that can clog the pores of the chromatographic medium, greatly hindering the immunochromatographic process. Other samples, such as blood, contain cells and colored components that make it difficult to read the results of the tests. Even if the sample does not create interference, it is frequently difficult with existing chromatographic test devices to apply the sample to the chromatographic medium so that the sample front moves uniformly through the chromatographic medium to ensure that the sample reaches the area where the binding is to occur in a uniform, straight-line manner.
Sample preparation and waste generation are responsible for other problems with currently available devices and techniques for immunochromatography. The increased prevalence of diseases spread by infected blood and blood fractions, such as AIDS and hepatitis, has exacerbated these problems. It is rarely possible to apply a sample (such as feces) or a sampling device (such as a throat swab) directly to the chromatographic medium. Several extraction and pretreatment reactions are usually required before the sample can be applied to the chromatographic medium. These reactions are typically carried out by the physician or technician performing the test in several small vessels, such as test tubes or microfuge tubes, requiring the use of transfer devices such as pipettes. Each of these devices is then contaminated and must be disposed of using special precautions so that workers or people who might inadvertently come into contact with the waste do not become contaminated and subject to infection by infectious agents contained in the waste.
Additionally, such a device should be capable of receiving a possibly contaminated sample or a sample preparation device directly so as to eliminate the need for extraction vessels and transfer devices. Such a device, preferably in the form of a test strip, should also be capable of performing immunochromatographic assays on colored samples or samples containing particulates without interference and should be able to deliver the sample to the chromatographic medium uniformly and evenly to improve accuracy and precision of the test. This aspect of an improved assay device is particularly important in avoiding false negatives and false positives.
Another aspect in which present immunochromatographic test devices need improvement is in reducing the volume of sample required to achieve the threshold sensitivity of the analyte to be determined. This is particularly important if the sample is whole blood and the donor is an infant, pediatric patient, geriatric patient, or patient who has suffered blood loss, limiting the volume of blood that can be drawn. If multiple tests are to be performed, as is often the case, it is crucial that the minimum volume of blood be used for each test. In addition, improvement is needed in allowing the more rapid performance of assays on immunochromatographic test strips to allow for the formation of a stable reaction endpoint in the shortest possible interval of time and to allow for a period where the color intensity of the reaction zones is stable for an additional period of time, allowing the operator to record an accurate result for a period of time, i.e., providing a stable reading window. This improves operator convenience and reduces the likelihood of error.
Another improvement in immunochromatographic assay test devices and formats is related to the use of monoclonal antibodies in assay test formats, particularly for assay of hCG. Although monoclonal antibodies can yield an increase in sensitivity and specificity for such assays, they can cause additional problems, particularly when murine monoclonal antibodies are used. If a plasma or serum sample is used, the human-anti-murine antibody (HAMA) response can cause interference in the assay, such as the occurrence of false negative or false positive results, when monoclonal antibodies are used.
Additionally, there is a need for assay devices that provide an accurate indication to the user that the assay has been completed. If the assay device is read before the assay has gone to completion, then an inaccurate result is very likely obtained. If the user cannot be certain when the reaction has been completed, the user is liable to wait an extended period of time, which is inefficient. Therefore, it would be desirable to have an assay device that would precisely indicate to the user when chromatography has occurred through the medium, so that the assay can be read and interpreted.
Additionally, it would be desirable to have an assay device that gives a semiquantitative indication of the concentration of the analyte assayed in a single test device so that a comparison can be made with a concentration standard. If a single test device cannot be used for such a comparison, a series of controls must be run in a number of separate test devices. It would therefore be desirable to have an assay device that can provide such a semi-quantitative indication in a single device with the use of a single sample.
Accordingly, there is a need for an assay device that gives a precise indication of the performance of the assay and for a device that can use monoclonal antibodies and control for the existence of HAMA. There is further a need for an immunochromatographic device that can efficiently assay a large number of analytes and utilize internal controls to indicate efficient performance of the assay. There is also a need for an improved immunochromatographic assay device that can give a semiquantitative indication of analyte concentration by comparison with a standard.